Decellularization of tissues and organs

Decellularized tissues and organs have been successfully used in a variety of tissue engineering/regenerative medicine applications, and the decellularization methods used vary as widely as the tissues and organs of interest. The efficiency of cell removal from a tissue is dependent on the origin of the tissue and the specific physical, chemical, and enzymatic methods that are used. Each of these treatments affect the biochemical composition, tissue ultrastructure, and mechanical behavior of the remaining extracellular matrix (ECM) scaffold, which in turn, affect the host response to the material. Herein, the most commonly used decellularization methods are described, and consideration give to the effects of these methods upon the biologic scaffold material.     

A novel chemical modification of bioprosthetic tissues using L-arginine

A novel chemical modification of biological tissues was developed by the direct coupling of bioactive molecule, L-arginine to bovine pericardium (BP). The modification involves pretreatment of BP using GA and followed by grafting arginine to BP by the reaction of residual aldehyde and amine group of L-arginine. BP was modified by direct coupling of bioactive molecules and the effect of L-arginine coupling on calcification and biocompatibility was evaluated in vitro and in vivo.Modified BPs were characterized by measuring shrinkage temperature, mechanical properties, digestion resistance to collagenase enzyme, in vitro plasma protein adsorption and platelet adhesion, and in vivo calcification. Thermal and mechanical properties showed that the durability of arginine treated tissue increased as compared with fresh tissue and GA treated tissue. Resistance to collagenase digestion revealed that modified tissues have greater resistance to enzyme digestion than did fresh tissue and GA treated tissue. Lower protein adsorption and platelet adhesion were observed on modified tissue than non-modified tissue. In vivo calcification study demonstrated much less calcium deposition on arginine treated BP than GA treated one. Obtained results attest to the usefulness of L-arginine treated BP for cardiovascular bioprostheses.     

Heparinized bovine pericardium as a novel cardiovascular bioprosthesis

A novel chemical modification of biological tissues was developed by the direct coupling heparin to bovine pericardium (BP). The heparinization involves pretreatment of BP using GA and followed by grafting heparin to BP by the reaction of residual aldehyde and amine group of heparin. BP was modified by direct coupling of heparin and the effect of heparin coupling on calcification was evaluated in vitro and in vivo. Heparinized BP was characterized by measuring shrinkage temperature, mechanical properties, digestion resistance to collagenase enzyme, in vitro cytotoxicity, and in vivo calcification. Thermal and mechanical properties showed that the durability of heparin-treated tissue increased as compared with fresh tissue and GA-treated tissue. Resistance to collagenase digestion revealed that heparin-treated tissue has greater resistance to enzyme digestion than did fresh tissue and GA-treated tissue. Heparinized tissue had shown to be non-cytotoxic, however, relatively high cytotoxicity was observed in the GA-treated tissues due to the release of GA. In vivo calcification study demonstrated much less calcium deposition on heparin-treated BP than GA-treated one. Obtained results attest to the usefulness of heparinized BP for cardiovascular bioprostheses.     

脱细胞技术在组织工程血管中的应用进展

将天然血管经过脱细胞处理得到的脱细胞血管,被认为是一种具有广阔应用前景的组织工程血管支架材料.截至目前,细胞外基质(ECM)支架的制备方法仍缺乏统一标准.脱细胞方法的选择取决于组织来源和基质支架的用途,尤其对于脱细胞血管等需要长期承受血流冲击的基质支架材料来说,脱细胞方案的选择至关重要.细胞清除效率和细胞外基质支架的性...     

Preservation of aortic root architecture and properties using a detergent-enzymatic perfusion protocol

Aortic valve degeneration and dysfunction is one of the leading causes for morbidity and mortality. The conventional heart-valve prostheses have significant limitations with either life-long anticoagulation therapeutic associated bleeding complications (mechanical valves) or limited durability (biological valves). Tissue engineered valve replacement recently showed encouraging results, but the unpredictable outcome of tissue degeneration is likely associated to the extensive tissue processing methods. We believe that optimized decellularization procedures may provide aortic valve/root grafts improved durability. We present an improved/innovative decellularization approach using a detergent-enzymatic perfusion method, which is both quicker and has less exposure of matrix degenerating detergents, compared to previous protocols. The obtained graft was characterized for its architecture, extracellular matrix proteins, mechanical and immunological properties. We further analyzed the engineered aortic root for biocompatibility by cell adhesion and viability in vitro and heterotopic implantation in vivo. The developed decellularization protocol was substantially reduced in processing time whilst maintaining tissue integrity. Furthermore, the decellularized aortic root remained bioactive without eliciting any adverse immunological reaction. Cell adhesion and viability demonstrated the scaffold's biocompatibility. Our optimized decellularization protocol may be useful to develop the next generation of clinical valve prosthesis with a focus on improved mechanical properties and durability.     

Tissue-derived biomaterials and their use in cardiovascular prosthetic devices

A variety of tissues and tissue components, ranging from allograft and xenograft tissues to albumin- and collagen-coated synthetic materials, have been used to fabricate cardiovascular prosthetic devices. Tissue-derived biomaterials include both viable and non-viable tissues as well as individual tissue components which have undergone some degree of preimplantation processing. A review of the biochemistry, immunogenicity, mechanical properties, physicochemical properties, preimplantation processing and the morphology of the following cardiovascular prostheses are discussed: heart valve bioprostheses and allografts; blood vessel allografts; biologic vascular grafts; and, protein-coated vascular prostheses. This review focuses on the generic aspects of prosthetic device fabrication using tissue-derived biomaterials, realizing that specific differences between various commercially available prostheses may exist.     

A mechanical evaluation of three decellularization methods in the design of a xenogeneic scaffold for tissue engineering the temporomandibular joint disc

Tissue-engineered temporomandibular joint (TMJ) discs offer a viable treatment option for patients with severe joint internal derangement. To date, only a handful of TMJ tissue engineering studies have been carried out and all have incorporated the use of synthetic scaffold materials. These current scaffolds have shown limited success in recapitulating morphological and functional aspects of the native disc tissue. The present study is the first to investigate the potential of a xenogeneic scaffold for use in tissue engineering the TMJ disc. The effects of decellularization agents on the disc's mechanical properties were assessed using three common decellularization protocols: Triton X-100, sodium dodecyl sulfate (SDS) and an acetone/ethanol solution. Decellularized scaffolds were subsequently characterized through cyclic mechanical testing at physiologically relevant frequencies to determine which chemical agent most accurately preserved the native tissue properties. Results have shown that porcine discs treated with SDS most closely matched the energy dissipation capabilities and resistance to deformation of the native tissue. Treatments using Triton X-100 caused the resultant tissue to become relatively softer with inferior energy dissipation capabilities, while treatment using acetone/ethanol led to a significantly stiffer and dehydrated material. These findings support the potential of a porcine-derived scaffold decellularized by SDS as a xenograft for TMJ disc reconstruction.     

Temporal evolution of skeletal regenerated tissue: what can mechanical investigation add to biological?

The objective here was to experimentally characterize the temporal evolution of the structural and mechanical properties of large volume immature regenerated tissues. We studied these evolving tissues from their genesis in controlled mechanical conditions. We developed an animal model based on the periosteal properties leading to unloaded regenerated skeletal tissue. To characterize the temporal evolution of mechanical properties, we carried out indentation tests coupled with macroscopic examinations and histological studies. This combined methodology yielded a range of information on osteogenesis at different scales: macroscopic by simple observation, mesoscopic by indentation test and microscopic by histological study. Results allowed us to identify different periods, providing a link between biological changes and material property evolution in bone tissue regeneration. The regenerated tissue evolves from a viscous, homogeneous, soft material to a heterogeneous stiffer material endowed with a lower viscosity. From a biological point of view, cell organization progresses from a proliferated cell clot to a mature structure closer to that of the bone. During the first 7 days, mechanical and biological results revealed the same evolution: first, the regenerated tissue grew, then, differentiated into an osteochondral tissue and finally calcification began. While our biological results confirm those of other studies, our mechanical results provide the first experimental mechanical characterization by reduced Young’s modulus of such tissue.     

An overview of tissue and whole organ decellularization processes

Biologic scaffold materials composed of extracellular matrix (ECM) are typically derived by processes that involve decellularization of tissues or organs. Preservation of the complex composition and three-dimensional ultrastructure of the ECM is highly desirable but it is recognized that all methods of decellularization result in disruption of the architecture and potential loss of surface structure and composition. Physical methods and chemical and biologic agents are used in combination to lyse cells, followed by rinsing to remove cell remnants. Effective decellularization methodology is dictated by factors such as tissue density and organization, geometric and biologic properties desired for the end product, and the targeted clinical application. Tissue decellularization with preservation of ECM integrity and bioactivity can be optimized by making educated decisions regarding the agents and techniques utilized during processing. An overview of decellularization methods, their effect upon resulting ECM structure and composition, and recently described perfusion techniques for whole organ decellularization techniques are presented herein.     

On the Decellularization of Fresh or Frozen Human Umbilical Arteries: Implications for Small-Diameter Tissue Engineered Vascular Grafts

Most tissues, including those to be decellularized for tissue engineering applications, are frozen for long term preservation. Such conventional cryopreservation has been shown to alter the structure and mechanical properties of tissues. Little is known, however, how freezing affects decellularization of tissues. The purpose of this study was two-fold: to examine the effects of freezing on decellularization of human umbilical arteries (HUAs), which represent a potential scaffolding material for small-diameter tissue-engineered vascular grafts, and to examine how decellularization affects the mechanical properties of frozen HUAs. Among many decellularization methods, hypotonic sodium dodecyl sulfate solution was selected as the decellularizing agent and tested on fresh HUAs to optimize decellularization conditions. The efficiency of decellularization was evaluated by DNA assay and histology every 12 up to 48 h. The optimized decellularization protocol was then performed on frozen HUAs. The stiffness, burst pressure, and suture retention strength of fresh HUAs and frozen HUAs before and after decellularization were also examined. It appeared that freezing decreased the efficiency of decellularization, which may be attributed to the condensed extracellular matrix caused by freezing. While the stiffness of fresh HUAs did not change significantly after decellularization, decellularization reduced the compliance of frozen HUAs. Interestingly, the stiffness of decellularized frozen HUAs was similar to that of decellularized fresh HUAs. Although little difference in stiffness was observed, we suggest avoiding freezing if more efficient and complete decellularization is desired.     

Altered structural and mechanical properties in decellularized rabbit carotid arteries

Recently, major achievements in creating decellularized whole tissue scaffolds have drawn considerable attention to decellularization as a promising approach for tissue engineering. Decellularized tissues are expected to have mechanical strength and structure similar to the native tissues from which they are derived. However, numerous studies have shown that mechanical properties change after decellularization. Often, tissue structure is observed by histology and electron microscopy, but the structural alterations that may have occurred are not always evident. Here, a variety of techniques were used to investigate changes in tissue structure and relate them to altered mechanical behavior in decellularized rabbit carotid arteries. Histology and scanning electron microscopy revealed that major extracellular matrix components were preserved and fibers appeared intact, although collagen appeared looser and less crimped after decellularization. Transmission electron microscopy confirmed the presence of proteoglycans (PG), but there was decreased PG density and increased spacing between collagen fibrils. Mechanical testing and opening angle measurements showed that decellularized arteries had significantly increased stiffness, decreased extensibility and decreased residual stress compared with native arteries. Small-angle light scattering revealed that fibers had increased mobility and that structural integrity was compromised in decellularized arteries. Taken together, these studies revealed structural alterations that could be related to changes in mechanical properties. Further studies are warranted to determine the specific effects of different decellularization methods on the structure and performance of decellularized arteries used as vascular grafts.     

Extraction techniques for the decellularization of tissue engineered articular cartilage constructs

Several prior studies have been performed to determine the feasibility of tissue decellularization to create a non-immunogenic xenogenic tissue replacement for bladder, vasculature, heart valves, knee meniscus, temporomandibular joint disc, ligament, and tendon. However, limited work has been performed with articular cartilage, and no studies have examined the decellularization of tissue engineered constructs. The objective of this study was to assess the effects of different decellularization treatments on articular cartilage constructs, engineered using a scaffoldless approach, after 4 wks of culture, using a two-phased approach. In the first phase, five different treatments were examined: 1) 1% SDS, 2) 2% SDS, 3) 2% Tributyl Phosphate, 4) 2% Triton X-100, and 5) Hypotonic followed by hypertonic solution. These treatments were applied for either 1 h or 8 h, followed by a 2 h wash in PBS. Following this wash, the constructs were assessed histologically, biochemically for cellularity, GAG, and collagen content, and biomechanically for compressive and tensile properties. In phase II, the best treatment from phase I was applied for 1, 2, 4, 6, or 8 h in order to optimize the application time. Treatment with 2% SDS for 1 h or 2 h significantly reduced the DNA content of the tissue, while maintaining the biochemical and biomechanical properties. On the other hand, 2% SDS for 6 h or 8 h resulted in complete histological decellularization, with complete elimination of cell nuclei on histological staining, although GAG content and compressive properties were significantly decreased. Overall, 2% SDS, for 1 or 2 h, appeared to be the most effective agent for cartilage decellularization, as it resulted in decellularization while maintaining the functional properties. The results of this study are exciting as they indicate the feasibility of creating engineered cartilage that may be non-immunogenic as a replacement tissue.     

Metal mesh scaffold for tissue engineering of membranes

Engineering of the membrane-like tissue structures to be utilized in highly dynamic loading environments such as the cardiovascular system has been a challenge in the past decade. Scaffolds are critical components of the engineered tissue membranes and allow them being formed in vitro and remain secure in vivo when implanted in the body. Several approaches have been taken to develop scaffolds for tissue membranes. However, all methods entail limitations due to structural vulnerability, short-term functionality, and mechanical properties of the resulted membrane constructs. To overcome these issues, we have developed a novel hybrid scaffold made of an extra thin layer of metal mesh tightly enclosed by biological matrix components. This approach retains all the advantages of using biological scaffolds while developing a strong extracellular matrix that can stand various types of loads after implantation inside the body.     

Decellularized bovine intervertebral disc as a natural scaffold for xenogenic cell studies

Low back pain that is associated with disc degeneration contributes to a huge economic burden in the worldwide healthcare system. Traditional methods, such as spinal fusion, have been adopted to relieve mechanical back pain, but this is compromised by decreased spinal motion. Tissue engineering has attracted much attention, and aims to correct the changes fundamentally occurring in the discs by a combination of cell biology, molecular biology and engineering. Synthetic materials including poly(l-lactic acid) or poly(glycolic acid) and biomolecules like hyaluronic acid or collagen have been adopted in the development of disc scaffolds for studying therapeutic approaches. Nevertheless, the complex biological and mechanical environment of the intervertebral disc (IVD) makes the synthesis of an artificial IVD with biomaterials a difficult task. Thus the aim of this study was to develop a natural disc scaffold for culturing disc cells for future development of biological disc constructs. We adopted a combination of currently used decellularization techniques to decellularize bovine IVD to create a complete endplate-to-endplate IVD scaffold. By altering the chemical and physical decellularization parameters, we reported the removal of up to 70% of the endogenous cells, and were able to preserve the glycosaminoglycan content, collagen fibril architecture and mechanical properties of the discs. The reintroduction of nucleus pulposus cells into the scaffold indicated a high survival rate over 7 days, with cell penetration. We have shown here that conventional methods used for decellularizing thin tissues can also be applied to large organs, such as IVD. Our findings suggest the potential of using decellularized IVD as a scaffold for IVD bioengineering and culturing of cells in the context of the IVD niche.     

Improved calcification resistance and biocompatibility of tissue patch grafted with sulfonated PEO or heparin after glutaraldehyde fixation

A novel chemical modification of biological tissues was developed aimed at improving biocompatibility and calcification resistance. This method involved the additional grafting of sulfonated PEO (PEO-SO(3)) or heparin after conventional glutaraldehyde (GA) fixation of bovine pericardium (BP). The amino groups of PEO-SO(3) or heparin were utilized to react to the GA residues to block them. The PEO-SO(3) or heparin grafted tissues demonstrated a slightly higher shrinkage temperature and tensile strength, but greater resistance to collagenase digestion, than GA treated ones. These results suggest that modified tissues have improved durability due to the grafting and filling effect of PEO-SO(3) or heparin in addition to the GA cross-linking. At the direct contact cytotoxicity test in vitro, PEO-SO(3) or heparin grafted tissue was shown to be nontoxic, while relatively significant cytotoxicity was observed for the GA treated tissues, possibly due to the release of GA. From the in vivo calcification study, calcium contents deposited on the modified tissues were much less than those on GA treated tissues. Such a decreased calcification might be explained by the decrease of residual GA groups during the additional treatment, and the space-filling effect and the nonadhesive property and/or the blood compatibility of PEO-SO(3) or heparin grafted covalently. The newly modified tissue patch was observed to show improved pathological assessibility including less inflammation and tissue reactions. This simple modification method may be useful for calcification-resistant and blood-compatible tissue patches for cardiovascular implants.     

The polyvinyl alcohol-bacterial cellulose system as a new nanocomposite for biomedical applications

Finding materials suitable for soft tissue replacement is an important aspect for medical devices design and fabrication. There is a need to develop a material that will not only display similar mechanical properties as the tissue it is replacing, but also shows improved life span, biocompatibility, nonthrombogenic, and low degree of calcification. Polyvinyl alcohol (PVA) is a hydrophilic biocompatible polymer with various characteristics desired for biomedical applications. PVA can be transformed into a solid hydrogel with good mechanical properties by physical crosslinking, using freeze-thaw cycles. Hydrophilic bacterial cellulose (BC) fibers of an average diameter of 50 nm are produced by the bacterium Acetobacter xylinum, using a fermentation process. They are used in combination with PVA to form biocompatible nanocomposites. The resulting nanocomposites possess a broad range of mechanical properties and can be made with mechanical properties similar to that of cardiovascular tissues, such as aorta and heart valve leaflets. The stress-strain properties for porcine aorta are matched by at least one type of PVA-BC nanocomposite in both the circumferential and the axial tissue directions. A PVA-BC nanocomposite with similar properties as heart valve tissue is also developed. Relaxation properties of all samples, which are important for cardiovascular applications, were also studied and found to relax at a faster rate and to a lower residual stress than the tissues they might replace. The new PVA-BC composite is a promising material for cardiovascular soft tissue replacement applications.     

Straining mode-dependent collagen remodeling in engineered cardiovascular tissue

Similar to native cardiovascular tissues, the mechanical properties of engineered cardiovascular constructs depend on the composition and quality of the extracellular matrix, which is a net result of matrix remodeling processes within the tissue. To improve tissue remodeling, and hence tissue mechanical properties, various mechanical conditioning protocols, such as strain-based or flow-based conditioning, have been applied to engineered cardiovascular constructs with promising results. We hypothesize that tissue remodeling is dependent on the mode of straining. Therefore, the effects of two modes of straining, being either static or dynamic, were quantified on several indices of tissue remodeling. Differences in matrix composition (collagen and glycosaminoglycans [GAGs]) and maturity (collagen cross-links) were quantified with time on gene expression and protein levels. In addition, the secretion of specific collagen remodeling markers (matrix metalloproteinase-1), collagen synthesis marker (procollagen type I carboxy-terminal propeptide, PIP), and collagen degradation marker (carboxyterminal telopeptide of type I, ICTP) was investigated with time. Static strain stimulated collagen gene expression and production with time. Dynamic straining resulted in (1) lower collagen gene expression and production, but (2) enhanced collagen cross-link expression and density, and GAG production, and (3) stimulated collagen remodeling processes, as expressed by enhanced production of remodeling markers. Thus, despite a lower collagen production, the quality of the neotissue was enhanced by a dynamic straining component. These straining mode-dependent remodeling responses allow us for the first time to balance collagen and cross-link production and, thus, to fine tune tissue mechanical properties via mechanical conditioning protocols. This is of utmost importance for cardiovascular tissue engineering, where insufficient mechanical properties are currently a main limiting factor for present in vivo application.     

改造天然生物组织为血管支架材料的预处理方法

目前 ,临床对血管替代物的需求量越来越大 ,传统的来源已不能满足需要。组织工程化血管的出现使得这一问题有望得到解决。构建组织工程化血管就必然涉及血管支架的预制。生物支架材料是血管支架中的一大类 ,它在细胞黏附及促细胞生长等方面优于人工合成的支架材料。由于生物性材料自活体取出后即开始降解 ,同时不同材料间也存在着种群差异 ,不宜保存和直接应用 ,故需采用一些预处理方法来解决这些问题 ,预处理目的就是在移植生物性材料前 ,降低其抗原性、提高其抗酶降解能力 ,并较长时间地保持其良好的力学性能和组织结构。这些处理方法包括运用戊二醛、多聚环氧化合物、碳化二亚胺、京尼平及原花色素等化学试剂进行交联的化学方法和应用光氧化等进行交联的物理方法。本文详细地叙述了各种预处理方法的机理及相应各种材料处理前后免疫原性、生物稳定性、力学性能、细胞毒性、抗钙化能力等特性的变化 ,并对各种方法的优缺点做一简要的评述。总之 ,生物性材料预处理的发展趋势是继续深入研究和开发细胞毒性小的天然交联剂 ,完善并拓宽光氧化交联的应用     

An in situ forming collagen-PEG hydrogel for tissue regeneration

There are limited options for surgeons to repair simple or complex tissue defects due to injury, illness or disease. Consequently, there are few treatments for many serious ailments, including neural-related injuries, myocardial infarction and focal hyaline cartilage defects. Tissue-engineered scaffolds offer great promise for addressing these wide-ranging indications; however, there are many considerations that need to be made when conceptualizing a product. For many applications, an in situ forming scaffold that could completely fill defects with complex geometries, adhere to adjacent tissues and foster cell proliferation would be ideal. Additionally, the scaffold would preferably have tailored mechanical properties similar to native tissues and highly controllable gelation kinetics, and would not require an external trigger, such as ultraviolet light, for gelation. We have developed a unique injectable hydrogel system composed of collagen and multi-armed poly(ethylene glycol) (PEG) that meets all of these criteria. The collagen component enables cellular adhesion and permits enzymatic degradation, while the multi-armed PEG component has amine-reactive chemistry that also binds proteins/tissue and is hydrolytically degradable. We have characterized the mechanical properties, swelling, degradation rates and cytocompatibility of these novel hydrogels. The hydrogels demonstrated tunable mechanics, variable swelling and suitable degradation profiles. Cells adhered and proliferated to near confluence on the hydrogels over 7 days. These data suggest that these collagen and PEG hydrogels exhibit the mechanical, physical and biological properties suitable for use as an injectable tissue scaffold for the treatment of a variety of simple and complex tissue defects.     

Glutaraldehyde-fixed kangaroo aortic wall tissue: histology,crosslink stability and calcification potential

Stentless aortic heart valve substitutes, manufactured from biological tissues, are fixed with glutaraldehyde to cross-link collagen, reduce antigenicity, and sterilize the tissue. Despite improved cross linking, reduced antigenicity, and various anticalcification measures, the aortic wall tissue present in these prostheses tends to calcify. The aim of this study was to assess the morphology, collagen cross-link stability, and calcification potential of glutaraldehyde-preserved kangaroo aortic wall tissue as opposed to porcine aortic wall tissue. Porcine and kangaroo aortic wall tissues were fixed in 0.625% buffered glutaraldehyde. Histology and cross-link stability were examined. Calcification potential was determined in the subcutaneous rat model. Kangaroo aortic wall tissue was significantly (p < 0.01) less calcified than porcine aortic wall tissue (26.67 +/- 6.53 versus 41.959 +/- 2.75 microg/mg tissue) at 8 weeks. In conclusion, the histological differences between kangaroo and porcine aortic wall tissue correlate well with the reduced calcification potential of kangaroo aortic wall tissue. The reduced calcification potential could result in improved long-term durability of stentless kangaroo heart valves as bioprostheses.